The present disclosure relates to a method of measuring the activity of an enzyme, and the like. More particularly, the disclosure relates to a method of measuring the activity of an enzyme by use of a multiple photon excitation process, and the like.
Measurement of enzymatic activities in a tissue or cell has been conducted by biochemical techniques. First, a part of a tissue is taken out to prepare a homogenate, or cultured cells are harvested to prepare a lysate. Next, the tissue homogenate or cell lysate is centrifuged to prepare an enzymatically active fraction. Then, a reagent to be a substrate for an enzyme is added to the enzymatic active fraction thus prepared, and the quantity of a substrate metabolite produced upon a reaction between the substrate reagent and the enzyme is measured, to thereby measure the enzymatic activity.
In a colorimetric method, for example, a substrate reagent capable of coloring by reaction with the enzyme is used, and the degree of coloring is determined to thereby measure the enzymatic activity. Other examples of the measuring method include a fluorometric method in which a substrate reagent showing fluorescence by reacting with an enzyme is used and the intensity of fluorescence is determined to thereby measure the enzymatic activity, and an absorptiometric method in which a substrate having an absorption wavelength varied through reaction with an enzyme is used and the variation in absorbance is determined to thereby measure the enzymatic activity.
Japanese Patent Laid-open No. 2006-34215 describes a preparation method for cytochrome P450 (hereinafter referred to as “CYP450”), which is a representative drug-metabolizing enzyme. Besides, Analytical Biochemistry 276:214-226, 1999 describes measurement of enzymatic activity by use of CYP450 obtained by this method or the like. In addition, American Society for Pharmacology and Experimental Therapeutics DMD 30:845-852, 2002 and Biochemistry 45:12204-12215, 2006 each disclose enzymatic activity measurement by use of CYP450 produced using a gene-recombined microorganism. Further, Japanese Patent Laid-open No. Hei 11-225791 describes enzymatic activity measurement in which a drug given to an animal is metabolized in vivo and the animal's urine is obtained to obtain CYP450.
In the methods of measuring an enzymatic activity according to the related art, the tissue homogenate or cell lysate and the enzymatically active fraction have to be prepared, which needs troublesome processes. Besides, in the methods of the related art, the tissue or cell has to be dissolved, so that it is very difficult to measure the activity, of an enzyme inside the tissue or cell while keeping the tissue or cell alive.
Thus, there is a need for a method of measuring an enzymatic activity in which measurement can be carried out by simpler operations, as compared with the methods according to the related art, and by which the activity of an enzyme present inside an in vivo tissue or an in vivo cell can also be measured.